The present invention relates to a process for removing a methionine (Met) residue at the N-terminus of peptides and proteins specifically.
Particularly the present invention relates to aminopeptidases which are derived from Bacillus licheniformis removes a methionine residue from the N-terminus of peptides and proteins. And the present invention relates to a process for preparing a natural type protein from recombinant proteins produced in microorganism. Various kinds of natural type proteins such as human growth hormone (HGH) can be prepared massively and easily by the process of the present invention.
Recombinant DNA technology permits large scale production of eukaryotic proteins in bacteria. However the proteins so produced are frequently characterized by the addition of an extra methionine residue at their N-terminus. The proteins containing the N-terminal methionine may induce immunogenic reaction in human and animal bodies and may not express effectively their original function. Therefore the process for preparing natural type proteins should be developed (Nature, 326, 315, 1987; J. Bacteriol., 169, 751-757, 1987; Bio/Technology, 8, 1036-1040, 1990; Appl. Microbiol. Biotechnol., 36, 211-215, 1991) to overcome the problems.
Especially human growth hormone (HGH) is a polypeptide composed of 191 amino acids, having 22, 125 kDa of molecular weight and containing Phe-Pro-Thr sequence at the N-terminus. And HGH is produced in human pituitary gland and used mostly to improve dwarfism (Raben, M. S., J. Clin. Endocr., 18, 901, 1958). Initially HGH has been extracted and purified from human pituitary gland to be utilized for medicinal treatment. However HGH has not been provided sufficiently to satisfy all the patients since the above process limits the HGH amount produced. And it is also reported that 4 children inoculated with HGH purified from hypothalamus have been infected with virus and died. Thus FDA has prohibited administration of HGH produced by the process for the treatment of patients (Science, 234, 22, 1986). Recently many researchers have attempted to produce HGH in Escherichia coli or yeast by using recombinant DNA technology and to utilize this HGH clinically (in E. coli, Korean Patent Publication No. 89-1244 and No. 87-701, Patent Laid-open No. 87-2258 and No. 84-8695; in yeast, Patent Publication No. 92-99 and Patent Laid-open No. 90-9973 and No. 90-9976).
Generally recombinant HGH has an extra methionine residue which is added at the N-terminus during protein synthesis by initiation codon although natural type HGH is composed of 191 amino acids without the methionine. Thus the recombinant HGH is produced as a form of methionyl HGH which is composed of 192 amino acids and contains Met-Phe-Pro-Thr sequence at the N-terminus. In respect of biological activity, methionyl HGH exhibits the same activity with that of natural type HGH (Moore, J. A., Endocrinology, 122, 2920-2926, 1988). Any other side-effect which an extra methionine induces has not been reported. But it is reported that antibodies against an extra methionine may be generated, of which the rate is higher than that of natural type proteins (Lancet, Mar. 29, 697, 1986).
Therefore, many researchers have attempted to produce natural type HGH without the N-terminal methionine. In particular a method has been developed to obtain natural type HGH in culture broth, which comprises fusing the N-terminus of HGH to a C-terminus of other proteins and cutting the fusion protein with specific protease (PCT WO 89/12678; EP 20209; EP 321940). And a method also has been attempted, which comprises expressing HGH in cell cytoplasm, secreting it out of cell an removing a methionine residue during secretion (EP 008832; U.S. Pat. No. 4,755,465; JP 01273591; EP 306673; Korean Patent Appl. No. 92-10932). Unfortunately these methods have some difficulties to perform complicated manipulation such as preparing expression vectors and transforming host cells. In addition fermentation condition should be optimized in each case.
And a method has been exploited in case of recombinant proteins containing an extra amino acid at the N-terminus, which uses aminopeptidases to remove the N-terminal amino acid and prepares natural type proteins by simple process. For example natural type HGH can be prepared from methionyl HGH when specific aminopeptidase removing the N-terminal methionine selectively is used. At that time methionyl HGH can be prepared in mass by the process already established (PCT WO 86/04609; WO 86/204527 A1).
Hitherto, several aminopeptidases which can be used to prepare natural type HGH have been reported, such as aminopeptidase purified from Aeromonas proteolytica (PCT WO 86/01229; EP 0489711 A3, B.T.G. Co.; Prescott and Wikes, Method in Enzymology, 44, 530-543, 1976), aminopeptidase from pig kidney (PCT 86.204527 A1; Bio/Technology, 5, 824-827, 1987, Takeda Co.), dipeptidyl aminopeptidase from Dictyostelium discoidem (EP 557076 A1; U.S. Pat. No. 5,126,249, Eli LiIly Co.) and aminopeptidase from Streptomyces thermonitrificans (EP 6296695; U.S. Pat. No. 5,569,598; Lucky Co.).
However the aminopeptidase should react with only extra amino acid residues of proteins at the N-terminus, not with naturally occurring sequences of amino acids in order to prepare natural type proteins. That is, when N-terminal amino acid sequences are X-Y-Z in natural type protein and Met-X-Y-Z in recombinant protein respectively, aminopeptidase which removes the methionine residue without reacting other amino acids, such as X-Y-Z-, is preferred to prepare the same protein with natural type proteins. Thus substrate specificity of aminopeptidase is important to satisfy the above conditions. Since methionyl HGH has Met-Phe-Pro-Thr-Ile amino acid sequence at the N-terminus, aminopeptidase for the use should recognize X-Pro sequence and stop cutting reaction in front of X amino acid to remove the N-terminal methionine selectively. For industrial use the enzyme also exhibits higher specific activity outstandingly.
Various kinds of aminopeptidase have been purified from microorganisms. Most of aminopeptidases require Ca2xe2x88x92, Zn2xe2x88x92 or other metal ions for their activities and remove amino acid residues at the N-terminus commonly. But aminopeptidases purified from different microorganisms have different enzymatic properties in respect of molecular weight, requirements of metal ions, optimal condition of reaction and substrate specificity (FEMS Microbiol. Rev., 18, 319-344, 1996). These aminopeptidases are classified into exo-peptidase removing amino acid from the N-terminus of substrate proteins gradually.
Presently aminopeptidases which have been purified from Bacillus genus are from Bacillus subtilis (Arch. Biochem. Biophys., 197, 63-77, 1979; Arch. Biochem. Biophys., 202, 540-545, 1980; J. Biochem., 107, 603-607, 1994; JP 03285684, Diacel-Chem Co.), Bacillus stearothermophilus (Meth. Enzymol., 19, 544-552, 1970; Biochem. Biophys. Acta, 438, 212-220, 1976; EP 101653, Unitika), Bacillus thuringensis (Biokhimiya, 49, 1899-1907, 1984), Bacillus licheniformis (Arch. Biochem. Biophys., 186, 383-391, 1978; Microbiol. Zh., 51, 49-52, 1989) and so forth.
In references cited above, aminopeptidases were analyzed to examine their enzymatic properties by using several substrates such as leucine-p-nitroanilide, dipeptide and the like. But oligopeptide and protein containing Met-X-Pro sequence at the N-terminus have not been used to examine aminopeptidases activity. Thus it has not been expected that aminopeptidases can be used for removal of only an extra methionine from recombinant proteins containing Met-X-Pro sequence at the N-terminus.
In order to prepare natural type proteins by using recombinant DNA technology, we have investigated and purified aminopeptidases of Bacillus licheniformis strains which remove only a methionine residue at the N-terminus of proteins. Aminopeptidases derived from Bacillus licheniformis an remove a methionine residue from synthetic substrate, oligopeptide, protein and so on properly and recognize Met-X-Pro sequence (X indicates any amino acid available) specifically. Thus we have demonstrated that aminopeptidases of the present invention can be used to prepare natural type HGH efficiently.
The object of the present invention is to provide aminopeptidases derived from Bacillus licheniformis which removes a methionine residue at the N-terminus of peptide and protein.
The aminopeptidases of the present invention recognize Met-X-Pro sequence at the N-terminus of peptide and protein to remove the methionine residue. Preferably X is phenylalanine.
The aminopeptidases contain amino acid sequences of SEQ ID. NO: 3 (Lys-Phe-Ser-Lys-Lys-Phe-Asn-Glu-Asn-Arg sequence), and SEQ ID. NO: 4 (Lys-Phe-Ser-Lys-Phe-Asn-Glu-Asn-Arg-Ala-Tyr-Gln-Tyr-Ile-Tyr-His-Leu sequence), at the N-terminus or sequence which are deleted and substituted in amino acids from the N-terminus of the above sequences.
The aminopeptidases have a molecular weight at the range of 43-47 kDa on SDS-PAGE.
The aminopeptidases are active at the range of pH 7.5-10.5 preferably and more preferably at the range of pH 8.5-9.5.
The aminopeptidases are active at the range of 40-70xc2x0 C. preferably and more preferably at the range of 50-60xc2x0 C.
The aminopeptidases are thermostable and maintains 50% activity even though it is incubated at 60xc2x0 C. for 4 hours.
The aminopeptidases activity are inhibited by dithiothreitol, iodoacetic acid and ortho-penanthroline and inhibited partially by ethylenediaminetetraacetic acid (EDTA).
Another object of the present invention is to provide processes for preparation of the aminopeptidases, Which comprise culturing Bacillus licheniformis, centrifuging the culture broth to obtain supernatant and performing gel filtration chromatography with the concentrated supernatant. Preferably Bacillus licheniformis strain is KCTC 3058, KCTC 12759, KCTC 3045 and KCTC 1030 in the above description.
The present invention provides a process for preparation of the aminopeptidases, which comprises preparing aminopeptidase-producing cells by using recombinant DNA technology, culturing the recombinant cells and purifying the enzyme.
Another object of the present invention is to provide a process for preparing natural type proteins, which uses recombinant proteins produced by using recombinant DNA technology with aminopeptidase and purifying proteins.
Precisely the present invention provides a process for preparing natural type HGH from methionyl HGH containing Met-X-Pro sequence at the N-terminus.
The process for preparation comprises using aminopeptidase at the range of pH 7.0-9.5 and at the range of 20-60xc2x0 C. and purifying natural type HGH through ion exchange resin chromatography and so forth. At that time NaCl is added to 30-230 mM into reaction mixture and enzymes is added at the range of 0.2-20 U per 1 mg of methionyl HGH preferably.